In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents. For example, immunotherapy, or the use of antibodies for therapeutic purposes has been used in recent years to treat cancer. Passive immunotherapy involves the use of monoclonal antibodies in cancer treatments. See for example, Cancer: Principles and Practice of Oncology, 6th Edition (2001) Chapt. 20 pp. 495-508. These antibodies can have inherent therapeutic biological activity both by direct inhibition of tumor cell growth or survival and by their ability to recruit the natural cell killing activity of the body's immune system. These agents can be administered alone or in conjunction with radiation or chemotherapeutic agents. Rituximab and Trastuzumab, approved for treatment of non-Hodgkin's lymphoma and breast cancer, respectively, are two examples of such therapeutics. Alternatively, antibodies can be used to make antibody conjugates where the antibody is linked to a toxic agent and directs that agent to the tumor by specifically binding to the tumor. Gemtuzumab ozogamicin is an example of an approved antibody conjugate used for the treatment of leukemia. Monoclonal antibodies that bind to cancer cells and have potential uses for diagnosis and therapy have been disclosed in publications. See, for example, the following patent applications which disclose, inter alia, some molecular weights of target proteins: U.S. Pat. No. 6,054,561 (200 KD c-erbB-2 (Her2), and other unknown antigens 40-200 KD in size) and U.S. Pat. No. 5,656,444 (50 KD and 55 KD, oncofetal protein). Example of antibodies in clinical trials and/or approved for treatment of solid tumors include: Trastuzumab (antigen: 180 kD, HER2/neu), Edrecolomab (antigen: 40-50 kD, Ep-CAM), Anti-human milk fat globules (HMFG1) (antigen >200 kD, HMW Mucin), Cetuximab (antigens: 150 kD and 170 kD, EGF receptor), Alemtuzumab (antigen: 21-28 kD, CD52), and Rituximab (antigen: 35 kD, CD20).
The antigen targets of trastuzumab (Her-2 receptor), which is used to treat breast cancer, and cetuximab (EGF receptor), which is in clinical trials for the treatment of several cancers, are present at some detectable level on a large number of normal human adult tissues including skin, colon, lung, ovary, liver, and pancreas. The margin of safety in using these therapeutics is possibly provided by the difference in the level of expression or in access of or activity of the antibody at these sites.
Another type of immunotherapy is active immunotherapy, or vaccination, with an antigen present on a specific cancer(s) or a DNA construct that directs the expression of the antigen, which then evokes the immune response in the individual, i.e., to induce the individual to actively produce antibodies against their own cancer. Active immunization has not been used as often as passive immunotherapy or immunotoxins.
Several models of disease (including cancer) progression have been suggested. Theories range from causation by a single infective/transforming event to the evolution of an increasingly “disease-like” or ‘cancer-like’ tissue type leading ultimately to one with fully pathogenic or malignant capability. Some argue that with cancer, for example, a single mutational event is sufficient to cause malignancy, while others argue that subsequent alterations are also necessary. Some others have suggested that increasing mutational load and tumor grade are necessary for both initiation as well as progression of neoplasia via a continuum of mutation-selection events at the cellular level. Some cancer targets are found only in tumor tissues, while others are present in normal tissues and are up-regulated and over-expressed in tumor tissues. In such situations, some researchers have suggested that the over-expression is linked to the acquisition of malignancy, while others suggest that the over-expression is merely a marker of a trend along a path to an increasing disease state.
One aspect required for the ideal diagnostic and/or therapeutic antibody is the discovery and characterization of an antigen that is associated with a variety of cancers. There are few antigens that are expressed on a number of types of cancer (e.g., “pan-cancer” antigen) that have limited expression on non-cancerous cells. The isolation and purification of such an antigen would be useful for making antibodies (e.g., diagnostic or therapeutic) targeting the antigen. An antibody binding to the “pan-cancer” antigen could be able to target a variety of cancers found in different tissues in contrast to an antibody against an antigen associated with only one specific type of cancer. The antigen would also be useful for drug discovery (e.g., small molecules) and for further characterization of cellular regulation, growth, and differentiation.
What is needed are novel targets on the surface of diseased and/or cancer cells that may be used to treat such diseases and/or cancers with antibodies and other agents which specifically recognize the cell surface targets. There exists a further need, based on the discoveries disclosed herein, for novel antibodies and other agents which specifically recognize targets on the surface of cells that can modulate, either by reducing or enhancing, the disease-promoting activities of EphA2.
EphA2, previously known as ECK, is a 130 kD transmembrane receptor tyrosine kinase that is expressed on adult epithelial cells. See Lindberg, et al. (1990) Mol. Cell. Biol. 10: 6316-6324. EphA2 is one member of the Eph family of receptor tyrosine kinases, which are unique in that they recognize ligands, known as ephrins, which are anchored to the membrane of adjacent cells. See Bartley, et al. (1994) Nature 368: 558-560. The sequence of the human receptor EphA2 is known in the literature. It encompasses an extracellular domain of 534 amino acids, a transmembrane domain of 24 amino acids, and a cytoplasmic domain of 418 amino acids that contains the tyrosine kinase domain.
EphA2 is over-expressed in a large number of cancer cells, for example, in breast, prostate, lung, and colon carcinomas, and in aggressive melanomas, but reportedly is not over-expressed in non-cancerous lesions of these same tissues. See, for example, Rosenberg, et al. (1997) Am. J. Physiol. 273: G824-G832; Easty, et al., (1995) Int. J. Cancer 60: 129-136; Walker-Daniels, et al. (1999) Prostate 41: 275-280; Zantek et al. (1999) Cell Growth & Differ. 10: 629-638; Zantek et al. (2001) Clin. Cancer Res. 7: 3640-3648; Zelniski et al. (2001) Cancer Res. 61: 2301-2306; WO 01/121172; and WO 01/12840. Moreover, cells that have been transformed to over-express EphA2 demonstrate malignant growth and ligand binding, which causes EphA2 to be internalized and degraded, and reverses the oncogenic effect of EphA2 over-expression. See Zelniski et al. (2001) Cancer Res. 61: 2301-2306; and Walker-Daniels, et al. (2002) Mol. Cancer. Res. 1: 79-87.
Using EphA2 as a therapeutic target has been proposed by others in the art. One author suggested using EphA2 ligands such as an Ephrin A1 fusion to human immunoglobulin G. Exposure of cells expressing EphA2 to the Ephrin A1-Fc fusion protein resulted in a down-regulation of EphA2 expression. See Duxbury, et al. (2004) BBRC 320:1096-1102.
Antibodies to eck/EphA2 are known, see e.g., Lindberg, et al. (1990) Mol. Cell. Biol. 10: 6316-6324. The use of antibody-based targeting using anti-EphA2 antibodies has been described by others, see e.g., Charles-Kinch et al. (2002) Cancer Res. 62:2840-2847, which describes using extracellular domain of EphA2 fused to human immunoglobulin to generated monoclonal antibodies against EphA2. Treatment of cancer cells with these anti-EphA2 monoclonal antibodies resulted in morphological changes and inhibition of cell growth on soft agar.
Antibodies to EphA2 have been made and proposed to be useful in the treatment of cancer (see e.g., U.S. Pat. No. 6,927,203; International Patent Publication Nos. WO01/12840 and WO01/12172; U.S. Provisional Patent Application Nos. 60/379,322 and 60/379,368; U.S. Pat. No. 5,824,303). WO2003US15044 describes methods comprising the administration of an effective amount of an antibody that binds to EphA2 and agonizes EphA2, thereby increasing EphA2 phosphorylation and decreasing EphA2 levels. In other embodiments, that application describes the administration of an effective amount of an antibody that binds to EphA2 and inhibits cancer cell colony formation in soft agar, inhibits tubular network formation in three-dimensional basement membrane or extracellular matrix preparation, preferentially binds to an EphA2 epitope that is exposed on cancer cells but not non-cancer cells, and has a low Koff, thereby, inhibiting tumor cell growth and metastasis. U.S. Pat. No. 6,927,203 describes antibodies that impede proliferation of tumor cells using an antibody that increases the phosphotyrosine content of EphA2.
PCT patent application WO 200391383 describes peptides derived from EphA2 and their use in anti-tumor immunotherapy. It describes peptide vaccination or immunotherapy based on an EphA2 epitope that may be used to induce or mimic a cytotoxic T lymphocyte cell response to tumor cells that over-express EphA2.
While EphA2 antibodies are known, there remains a need for particular anti-EphA2 antibodies that are extremely effective in inhibiting tumor cell growth, beyond the level of effectiveness shown by the EphA2 antibodies shown in the prior art.
All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.